Metagenomic Analysis of Bacterial and Fungal Communities Inhabiting Shiro Dominant Soils of Two Production Regions of Tricholoma Matsutake S

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Article Metagenomic Analysis of Bacterial and Fungal Communities Inhabiting Shiro Dominant Soils of Two Production Regions of Tricholoma Matsutake S. Ito & S. Imai in Korea

Gi-Hong An , Jae-Han Cho, Ok-Tae Kim and Jae-Gu Han *

Mushroom Research Division, National Institute of Horticultural and Herbal Science, RDA, Eumseong, Chungbuk 27709, Korea; [email protected] (G.-H.A.); [email protected] (J.-H.C.); [email protected] (O.-T.K.) * Correspondence: [email protected]; Tel.: +82-43-872-5732

Abstract: Tricholoma matsutake is an ectomycorrhizal fungus that has obligate symbiotic relationships with Pinus densiflora. Its fruiting body has a distinctive flavor and is traded at a high price. Thus, it has been a significant source of income for rural communities in Korea. We hypothesized that biotic factors considerably influence the formation of the T. matsutake mushroom, and the soils producing T. matsutake share similar microbial characteristics. Therefore, the present study aimed to detect the specific fungal and bacterial groups in T. matsutake production soils (shiro+) and nonproduction soils (shiro−) of the Bonghwa and Yanyang regions via next-generation sequencing. In a total of 15 phyla, 36 classes, 234 genera of bacteria, six phyla, 29 classes, and 164 genera of fungi were detected from four samples at both sites. The species diversity of shiro+ soils was lower than the shiro− samples in both the fungal and bacterial groups. In addition, we did not find high similarities in the microbial communities between the shiro+ soils of the two regions. However, in the resulting differences Citation: An, G.-H.; Cho, J.-H.; Kim, between the fungal communities categorized by their trophic assembly, we found a distinguishable O.-T.; Han, J.-G. Metagenomic compositional pattern in the fungal communities from the shiro+ soils and the shiro− soils of the Analysis of Bacterial and Fungal Communities Inhabiting Shiro two sites. Thus, the similarity among the microbial communities in the forest soils may be due to Dominant Soils of Two Production the fact that the microbial communities in the T. matsutake dominant soils are closely associated with Regions of Tricholoma Matsutake S. Ito biotic factors and abiotic factors such as soil properties. & S. Imai in Korea. Forests 2021, 12, 758. https://doi.org/10.3390/ Keywords: ectomycorrhizae; bacterial communities; fungal communities; metagenomics; miseq; f12060758 shiro dominant soil; Tricholoma matsutake

Academic Editor: Carol A. Loopstra

Received: 26 April 2021 1. Introduction Accepted: 4 June 2021 Tricholoma matsutake (S. Ito & S. Imai) that forms a symbiotic association with the Published: 9 June 2021 root tips of Pinus densiflora (Siebold & Zucc.) provides attractive commercial benefits to rural communities in Korea [1,2]. The annual yields of this mushroom are highly Publisher’s Note: MDPI stays neutral limited and unpredictable. Since it has not yet been successfully artificially cultivated, the with regard to jurisdictional claims in entire production of T. matsutake still depends upon natural harvesting from forests. In published maps and institutional affil- recent decades, many researchers have strived to succeed in the artificial production of iations. T. matsutake [2–6]. However, the artificial cultivation of this fungus has not been established. As an obligated symbiont, the biology of this mycorrhizal fungus must be considered from the perspective of the ecological interaction with the surrounding biotic factors, especially microbial groups. Copyright: © 2021 by the authors. Soil ecosystems have a wide variety of microbial communities. Microorganisms in Licensee MDPI, Basel, Switzerland. the soil can have positive or negative effects on the growth of ectomycorrhizal fungus [7]. This article is an open access article Many studies have been conducted on the microbial communities in the soils adjacent distributed under the terms and to T. matsutake [6,8–11]. The influence of the diverse microbial communities in soil on conditions of the Creative Commons the life cycle of T. matsutake, such as the development of mycelia and the formation of Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ fruiting bodies in various ways, has been investigated [4,12,13]. In particular, some soil 4.0/). bacteria, which are called mycorrhizal helper bacteria (MHB), have beneficial effects on the

Forests 2021, 12, 758. https://doi.org/10.3390/f12060758 https://www.mdpi.com/journal/forests Forests 2021, 12, 758 2 of 16

mycorrhizal symbiosis by mobilizing nutrients in nutrient-deficient soils [14]. In addition, some fungi that have frequently been detected from the fruiting body and fairy ring of T. matsutake co-exist in the hyphal dominant environment as a potential mycorrhizal helper fungus [11]. Therefore, the various microbial communities associated with T. matsutake could potentially contribute to the growth of hyphae and the formation of the fruiting body of T. matsutake [7,15–17]. Recent advances in metagenomics contribute to unveiling the microbial communities in various environmental samples [11,13,18]. Next-generation sequencing (NGS) based on metagenomics is a culture-independent method that enables the identification of uncultured microbes. The recent application of NGS sequencing methods, such as pyrosequencing 454 and Illumina, may provide a more direct way to detect microbial taxa, especially those with a low level of species changes [19,20]. In addition, Illumina sequencing is cost-effective and could obtain tenfold or more sequences per sample than pyrosequencing 454, thereby allowing the analysis of a high number of detailed taxonomic profiles from samples [21]. According to the report by Korean Statistical Information Service (KOSIS), the annual yields of T. matsutake in Korea dramatically decreased from 480 tons in 2007 to 140 tons in 2017. Previously, Gyeongsangbuk-do and Gangwon-do provinces were well-known as the representative T. matsutake producing regions that occupied the largest (61.3%) and second (18.0%) largest total yields of T. matsutake in Korea. However, the yields of T. matsutake in Gyeongsangbuk-do province have been gradually decreasing, and it was reported by KOSIS to be 340 tons in 2007, further decreasing to 75 tons in 2017. Thus, we speculated that the reduction in the yields of T. matsutake is due to various environmental factors, and we focused on the changes in microbial communities in the production of soils for T. matsutake. We hypothesized that the microbial communities in the production of soils for T. matsutake would be quite similar to one another, regardless of geographical characteristics, if there are microorganisms that have beneficial effects on the growth of mycelium and the formation of the fruit body of T. matsutake. Therefore, in this study, to determine the differences between the microbial communities in soils where T. matsutake occurs in Gyeongsangbuk- do and Gangwon-do provinces, we conducted sampling of the T. matsutake production soil (shiro+ soil) and nonproduction soil (shiro− soil) in two main T. matsutake production regions (Bonghwa and Yangyang) in Korea and investigated the soil bacterial and fungal communities from each sample using the Illumina Miseq sequencing platform

2. Materials and Methods 2.1. Sampling Sites Two sampling sites were mountains located near Seo-myeon, Yangyang-gun, Gangwon- do (110 m ELV, 38◦03023.8” N, 128◦38038.7” E), and Beopjeon-myeon, Bonghwa-gun, Gyeongsangbuk-do (360 m ELV, 36◦55014.2” N, 128◦56047.2” E) in south-eastern parts of the Korean peninsula. The climate and major vegetation at these sites are summarized in Table1. At the Yangyang site, in 2019, the annual mean temperature was 13.9 ◦C, and annual precipitation was 1517.3 mm. The major canopy vegetation was P. densiﬂora at more than 80%, and understory vegetation comprised Rhododendron schlippen (Maxim.), R. mucronulatum (Turcz.), Smilax nipponica (Miq.), and Carex fernaldiana (H.Lév & Vaniot). The site belongs to the public research forest under the management of the Yangyang-gun Agricul- tural Technology Center. At the Bonghwa site, in 2019, the annual mean temperature was 11.5 ◦C, and annual precipitation was 970.5 mm. The major canopy vegetation was P. densi- ﬂora at more than 80%, and understory vegetation comprised R. schlippen, R. mucronulatum, Melampyrum roseum (Maxim.), Pteridium aquilinum (Underw. ex A. Heller), S. nipponica, and C. fernaldiana. The site in Bonghwa is privately owned. The sampling sites are geographi- cally remote, located approximately 120 km from each other (Figure1). In September 2019, soil sampling was conducted immediately after T. matsutake fruiting bodies were harvested. By using a soil sampler, soils containing shiro (shiro+) were collected at 10 cm depth from the soil surface at the three spots of the zone beneath the T. matsutake, and soils without Forests 2021, 12, x FOR PEER REVIEW 3 of 17

September 2019, soil sampling was conducted immediately after T. matsutake fruiting bodies were harvested. By using a soil sampler, soils containing shiro (shiro+) were collected Forests 2021, 12,at 758 10 cm depth from the soil surface at the three spots of the zone beneath the T. matsutake, 3 of 16 and soils without shiro (shiro−) were collected at approximately 3–4 m intervals from the spots with the shiro+ soils. Each soil sample was placed into a polyvinyl bag and mixed well. Shiro can beshiro distinguished (shiro−) wereby its collectedfeatures of at whitish-gray-colored approximately 3–4 m soil, intervals in which from fun- the spots with the gal hyphae are aggregatedshiro+ soils. [11]. Each Soil soil samples sample taken was placedfrom each into sampling a polyvinyl site bag were and trans- mixed well. Shiro can ported on ice andbe stored distinguished at −4 °C before by its DNA features extraction. of whitish-gray-colored soil, in which fungal hyphae are aggregated [11]. Soil samples taken from each sampling site were transported on ice and Table 1. Climates andstored vegetation at −4 ◦ofC the before experime DNAntal extraction. sites (http://www.nongsaro.go.kr/). Temp./Precipitation Major Vegetation Table 1. ClimatesSites and vegetationLocation of the experimental sites (http://www.nongsaro.go.kr/, (accessed on 26 April 2021)). (Elevation) (Canopy/Understory) Yangyang Gangwon-do 13.9Temp./Precipitation °C/1517.5 mm Pinus densiflora SieboldMajor & Vegetation Zucc./ Sites Location (Elevation) Rhododendron schlippen(Canopy/Understory) Maxim. Yangyang Gangwon-do 13.9 ◦C/1517.5 mm R. mucronulatumPinus densiﬂora TurczSiebold. & Zucc./ (160 m ELV.) Smilax nipponicaRhododendron Miq schlippen. Maxim. R. mucronulatum Turcz. (160 m ELV.) Carex fernaldiana H.Lév & Vaniot Smilax nipponica Miq. Bonghwa Gyeonsangbuk-do 11.5 °C/970.5 mm PinusCarex densiflora/ fernaldiana H.Lév & Vaniot Bonghwa Gyeonsangbuk-do (360 11.5 m◦C/970.5 ELV.) mm Rhododendron schlippenPinus densiﬂora/ (360 m ELV.) R. mucronulatumRhododendron schlippen Melampyrum roseumR. mucronulatumMaxim. Melampyrum roseum Maxim. PteridiumPteridium aquilinum aquilinum Underw.Underw. ex ex A. Heller A. HellerSmilax nipponica Smilax nipponicaCarex fernaldiana Carex fernaldiana

Figure 1. Sampling sites were located in Yangyang-gun, Gangwon-do (A), and Bonghwa-gun, Figure 1. Sampling sites were located in Yangyang-gun, Gangwon-do (A), and Bonghwa-gun, Gyeongsangbuk-do (B) Gyeongsangbuk-do (B) in Korea. The map was downloaded from National Geographic Infor- in Korea. The map was downloaded from National Geographic Information Institute (NGII, https://www.ngii.go.kr/ mation Institute (NGII, https://www.ngii.go.kr/(11 June 2020)). (accessed on 11 June 2020)).

2.2. DNA Extraction, Library Construction, and Illumina Miseq Sequencing For each sample, microbial DNA was extracted from 0.5–1 g per soil using a DNeasy Power Soil Kit (Qiagen, Hilder, Germany) according to the manufacturer’s instructions. The extracted DNA was quantiﬁed using Quant-IT PicoGreen (Invitrogen). The sequencing libraries were prepared according to the Illumina 16S Metagenomic sequencing library protocols for the V3–V4 region for bacteria, and 5.8S and ITS2 regions for fungi. For bacteria, Forests 2021, 12, 758 4 of 16

the input 2 uL (10 ng uL−1) was PCR amplified with 1× reaction buffer, 1 nM dNTP mix, 500 nM concentrations of the universal F/R PCR primers, and 2.5 U of Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA, USA). The cycle condition for the 1st PCR was 3 min at 95 ◦C for heat activation, and 25 cycles of 30 s at 95 ◦C, 30 s at 55 ◦C, and 30 s at 72 ◦C, followed by a 5 min final extension at 72 ◦C, The 1st PCR product was purified with AMPure beads (Agencourt Bioscience, Beverly, MA, USA). Following purification, the 2 uL of 1st PCR product was PCR amplified for final library construction containing the index using the NexteraXT Indexed Primer. The cycle condition for the 2nd PCR was the same as the 1st PCR condition except for 10 cycles. The PCR product was purified with AMPure beads. The resulting PCR products were pooled, and the fragment sizes were checked using agarose gel electrophoresis and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The final purified product was then quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification Kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). The paired-end (2 × 300 bp) sequencing was performed by the Macrogen using the MiSeq™ platform (Illumina, San Diego, CA, USA).

2.3. Processing and Analyzing of Sequencing Data The bacterial and fungal sequence reads were assembled using FLASH 1.2.11 (Fast Length Adjustment of Short reads, http://ccb.jhu.edu/software/FLASH/ (accessed on 17 December 2019)). After assembly, pre-processing and clustering were carried out using the CD-HIT-OTU program (http://weizhongli-lab.org/cd-hit-otu/ (accessed on 17 Decem- ber 2019)) that performed the OTU (Operational Taxonomic Units) finding. CD-HIT-OTU comprises the following steps: (1) Low-quality reads are filtered out and extra-long tails are trimmed. (2) Filtered reads are clustered at 100% identity using CD-HIT-DUP. (3) Chimeric reads are identified. (4) Secondary clusters are recruited into primary clusters. (5) Noise sequences in clusters of size x or below are removed. Size x is statistically calculated. (6) Remaining representative reads from non-chimeric clusters are clustered into OTUs at a user-specified OTU cutoff (e.g., 97% identity at species level) [22]. Representative sequences for each OUT were selected and assigned to taxonomic data at RDP for bacteria [23] and UNITE for fungi [24] databases using the Quantitative Insights Into Microbial Ecology (QIIME) which is an open-source bioinformatics pipeline for performing microbiome analysis from raw DNA sequencing data [25]. Alpha diversity indices, such as the Shannon index, Chao 1, Simpson index, and Good’s coverage were also calculated using QIIME [25]. Sorensen’s classic similarity analysis that is based on the probability between two randomly chosen individuals, one from each of the two samples, was performed using EstimateS 9.1.0 [26].

3. Results 3.1. Statistical Data Analysis for Bacterial Communities in Sampling Sites A total of 473,975 reads and 1315 OTUs were detected in four sampling spots (Table2 ). The number of total reads ranged from 112,489 to 129,607. In all, 221 to 379 OTUs per sampling spot were obtained at a 99% similarity level. The result of the Chao1 estimation showed that the species richness of the shiro− soil samples of the Bonghwa and Yangyang sites was lower than that of the shiro+ soils. The diversity of species of bacterial communities for each sampling spot indicated that the shiro− soils included more bacterial communities than shiro+ soils at both sampling sites. The shiro− soil at the Bonghwa site represented the most diverse bacterial community (Shannon index = 6.777), while the shiro+ soil of Bonghwa had the lowest bacterial diversity (4.628). The Good’s coverage of all sampling spots ranged from 0.997 to 0.999, indicating that the sequencing depth appropriately represented the bacterial diversity. The rarefaction curves, which reveal the species richness of each sample, also showed that the shiro− soil samples of both of the sampling sites have a greater bacterial community than the shiro+ soil samples (Figure2a). Forests 2021, 12, 758 5 of 16

Table 2. Summary of Illumina sequencing and statistical analysis of bacterial communities inhabiting the Tricholoma matsutake production (shiro+) and nonproduction (shiro−) soils of Bonghwa (B) and Yangyang (Y) sampling sites.

B_Shiro+ B_Shiro− Y_Shiro+ Y_Shiro− Number of total reads 115,236 116,643 112,489 129,607 Number of OTUs 340 375 221 379 Chao1 estimation 349.5 385.1 230.0 400.7 Shannon index 4.628 6.777 5.187 6.212 Inverse simpson index 0.885 0.977 0.937 0.969 Forests 2021, 12, x FOR PEER REVIEW 6 of 17 Good’s coverage 0.999 0.997 0.999 0.997

(a) (b)

FigureFigure 2. 2.RarefactionRarefaction curves curves for for chao1 chao1 of ofbacteria bacteria (a) ( aand) and fungi fungi (b ()b from) from soil soil samples samples inhabiting inhabiting the the TricholomaTricholoma matsutake matsutake productionproduction (shiro+) (shiro+) and and nonproduction nonproduction (shiro (shiro−) −soils) soils of Bonghwa of Bonghwa and and Yangyang Yangyang sampling sampling sites. sites.

3.3. Relative Abundance of Bacterial Communities 3.2. Statistical Data Analysis for Fungal Communities in Sampling Sites Taxonomic composition analysis of bacteria for four sampling spots was conducted A total of 496,994 reads and 781 OTUs were detected at four sampling spots (Table3 ). at Forthe thephylum number level of (Figure OTUs, the3a). shiro In total,− soil 15 sample phyla were of Bonghwa identified: was 12 the phyla highest in the (364 shiro+ OTUs ), soilfollowed of Bonghwa, by the shiro10 phyla− soil in of the Yangyang shiro− (244soil OTUs),of Bonghwa, the shiro+ 12 phyla soil of in Yangyang the shiro+ (107 soil OTUs of ), Yangyang,and the shiro+ and 13 soil phyla of Bonghwain the shiro (66− soil OTUs). of Yangyang. All indices At ofthe Chao1, Bonghwa Shannon site, Bacteroidetes diversity, and andinverse Proteobacteria Simpson werein the lowershiro+ in soil the were shiro relatively− soil than abundant the shiro+ (46.01% soil samples and 45.08%), in both com- sam- paredpling with sites. the In shiro particular,− soil (14.56% the fungal and diversity 38.16%). of Acidobacteria the shiro− soil was sample the dominant of Bonghwa phylum (Chao1 in estimationthe shiro− =soil 364; (37.92%) Shannon compared index = 5.846;with the Inverse shiro+ Simpson soil (3.44%). index =At 0.948) the Yangyang was the highest site, theamong highest the abundance soil samples. of phyla All samplesin the shiro+ showed soil was Good’s shown coverage by Actinobacteria, that indicated and sufﬁcient Chlor- oflexisequencing compared depth with for those characterizing of the shiro fungal− soil, whereas diversity. Acidobacteria, The rarefaction Proteobacteria, curves also showed and Verrucomicrobiathat the shiro− weresoil samples the dominant of both phyla of the in sampling the shiro sites− soil. have a higher number of OTUs thanThe the relative shiro+ soilabundance samples at of boththe class sites (Figurelevel for2b). Acidobacteria, Actinobacteria, Bac- teroidetes, and Proteobacteria, the most frequently observed phyla in the four soil samples, showed distinct differences between the soil samples and the sampling sites. Of the total 36 classes, four classes of Acidobacteria phylum, four classes of Actinobacteria phylum, four classes of Bacteroidetes phylum, and five classes of Proteobacteria phylum were mainly observed. Acidobacteria, Acidobaceriia, and Vicinamibacteria were commonly detected in all soil samples (Figure 3b). Within the phylum Actinobacteria, the most abundant class in all samples was Actinobacteria (Figure 3c). Chitinophagia and Sphingobac- teriia commonly showed Bacteroidetes in all soil samples (Figure 3d). Five bacterial classes of Proteobacteria phylum, i.e., Alphaproteobacteria, Betaproteobacteria, Deltaproteo- bacteria, Gammaproteobacteria, and Oligoflexia, were commonly detected in all samples (Figure 3e). The Alphaproteobacteria class was highly detected in the shiro− soils of both sites, while the Betaproteobacteria class was more abundant in the shiro+ soils than the shiro− soils at both sites.

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Table 3. Summary of Illumina sequencing and statistical analysis of fungal communities inhabiting the Tricholoma matsutake production (shiro+) and nonproduction (shiro−) soils of Bonghwa (B) and Yangyang (Y) sampling sites.

B_Shiro+ B_Shiro− Y_Shiro+ Y_Shiro− Number of total reads 142,261 113,784 127,090 113,859 Number of OTUs 66 364 107 244 Chao1 estimation 66.5 364 111.7 246.1 Shannon index 1.355 5.846 1.873 3.845 Inverse simpson index 0.498 0.948 0.505 0.840 Good’s coverage 1.000 1.000 1.000 1.000

3.3. Relative Abundance of Bacterial Communities Taxonomic composition analysis of bacteria for four sampling spots was conducted at the phylum level (Figure3a). In total, 15 phyla were identiﬁed: 12 phyla in the shiro+ soil of Bonghwa, 10 phyla in the shiro− soil of Bonghwa, 12 phyla in the shiro+ soil of Yangyang, and 13 phyla in the shiro− soil of Yangyang. At the Bonghwa site, Bacteroidetes and Proteobacteria in the shiro+ soil were relatively abundant (46.01% and 45.08%), compared with the shiro− soil (14.56% and 38.16%). Acidobacteria was the dominant phylum in the shiro− soil (37.92%) compared with the shiro+ soil (3.44%). At the Yangyang site, the highest abundance of phyla in the shiro+ soil was shown by Actinobacteria, and Forests 2021, 12, x FOR PEER REVIEW 7 of 17 Chloroﬂexi compared with those of the shiro− soil, whereas Acidobacteria, Proteobacteria, and Verrucomicrobia were the dominant phyla in the shiro− soil.

FigureFigure 3. Taxonomic 3. Taxonomic composition composition analysis analysis of bacterial of bacterial communities communities at the atthe phylum phylum level level (a) (anda) and class class level level (b– (eb) –inhibitinge) inhibiting thethe TricholomaTricholoma matsutake matsutake productionproduction (shiro+) (shiro+) and and nonproduction nonproduction (shiro (shiro−) soils−) soils of Bonghwa of Bonghwa and and Yangyang Yangyang sampling sampling sites. sites. (b) (fourb) four bacterial bacterial classes classes of ofAcidobacteria Acidobacteria phylum, phylum, (c) ( c4) bacterial 4 bacterial classes classes of of Actinobacteria Actinobacteria phylum, phylum, (d (d) )4 4 bacterial bacterial classes classes of of BacteroidetesBacteroidetes phylum, phylum, and and ( (ee)) 5 5 bacterial bacterial classes classes of of Proteobacteria Proteobacteria phylum.

In theThe total relative 234 genera abundance of bacteria, at the 42 class genera level were for observed Acidobacteria, in more Actinobacteria, than 1% of rela- Bac- tiveteroidetes, abundance and in Proteobacteria,the shiro+ soils the and most shiro frequently− soils of observedthe two sampling phyla in thesites four (Table soil 4). samples, Of theseshowed genera, distinct Mucilaginibacter differences between (26.19%) the from soil samplesBacteriodetes and the phylum, sampling Acidobacterium sites. Of the total (11.46%) from Acidobacteria phylum, Actinoallomurus (18.17%) from Actinobacteria phylum, and Mycobacterium (14.50%) from Actinobacteria phylum were the most dominant genera in the shiro+ soil of Bonghwa, the shiro− soil of Bonghwa, the shiro+ soil of Yang- yang, and the shiro− soil at the Yangyang site, respectively. Seven genera, Flavobacterium (4.44%), Pedobacter (2.92%), Sphingobacterium (8.56%), Novosphingobium (1.60%), Janthino- bacterium (21.94%), Pseudomonas (6.73%), and Stenotrophomonas (1.76%), were only observed in the shiro+ soil at the Bonghwa site. In addition, the genera of Acidipila (2.98%), Silvibacterium (1.28%), Labedaea (13.37%), Pseudonocarida (1.16%), Sinosporangium (1.95%), Actinoallomurus (18.17%), Actinocorallia (1.79%), Actinomadura (3.91%), Dictyobacter (8.63%), Rhodopila (3.58%), Aliidongia (1.98%), and Caballeronia (2.18%) were more abundantly detected in the shiro+ soil at the Yangyang site among the four samples. The only two genera that were observed with high abundances in the shiro+ soils of the two sites were Caballeronia, and Paraburkholderia, while eight genera, Acidobacterium, Edaphobacter, Granulicella, Paludibaculum, Phenylobacterium, Rhizomicrobium, Sulfuriflexus, and Povalibac- ter, have high proportions in the shiro− soils of the two sites.

Table 4. List of bacterial genera inhabiting the Tricholoma matsutake production (shiro+) and nonproduction (shiro−) soils of Bonghwa and Yangyang sampling sites. The bacterial genera present more than 1% of at least one sample among the four soil samples.

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36 classes, four classes of Acidobacteria phylum, four classes of Actinobacteria phylum, four classes of Bacteroidetes phylum, and five classes of Proteobacteria phylum were mainly observed. Acidobacteria, Acidobaceriia, and Vicinamibacteria were commonly detected in all soil samples (Figure3b). Within the phylum Actinobacteria, the most abundant class in all samples was Actinobacteria (Figure3c). Chitinophagia and Sphingobacteriia commonly showed Bacteroidetes in all soil samples (Figure3d). Five bacterial classes of Proteobacteria phylum, i.e., Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and Oligoflexia, were commonly detected in all samples (Figure3e ). The Alphaproteobacteria class was highly detected in the shiro− soils of both sites, while the Betaproteobacteria class was more abundant in the shiro+ soils than the shiro− soils at both sites. In the total 234 genera of bacteria, 42 genera were observed in more than 1% of relative abundance in the shiro+ soils and shiro− soils of the two sampling sites (Table4). Of these genera, Mucilaginibacter (26.19%) from Bacteriodetes phylum, Acidobacterium (11.46%) from Acidobacteria phylum, Actinoallomurus (18.17%) from Actinobacteria phylum, and Mycobacterium (14.50%) from Actinobacteria phylum were the most dominant genera in the shiro+ soil of Bonghwa, the shiro− soil of Bonghwa, the shiro+ soil of Yangyang, and the shiro− soil at the Yangyang site, respectively. Seven genera, Flavobacterium (4.44%), Pedobacter (2.92%), Sphingobacterium (8.56%), Novosphingobium (1.60%), Janthinobacterium (21.94%), Pseudomonas (6.73%), and Stenotrophomonas (1.76%), were only observed in the shiro+ soil at the Bonghwa site. In addition, the genera of Acidipila (2.98%), Silvibacterium (1.28%), Labedaea (13.37%), Pseudonocarida (1.16%), Sinosporangium (1.95%), Actinoallomu- rus (18.17%), Actinocorallia (1.79%), Actinomadura (3.91%), Dictyobacter (8.63%), Rhodopila (3.58%), Aliidongia (1.98%), and Caballeronia (2.18%) were more abundantly detected in the shiro+ soil at the Yangyang site among the four samples. The only two genera that were observed with high abundances in the shiro+ soils of the two sites were Caballeronia, and Paraburkholderia, while eight genera, Acidobacterium, Edaphobacter, Granulicella, Paludibacu- lum, Phenylobacterium, Rhizomicrobium, Sulfuriflexus, and Povalibacter, have high proportions in the shiro− soils of the two sites.

Phylum Family Genus B_Shiro+ B_Shiro− Y_Shiro+ Y_Shiro− Acidobacteria Acidobacteriaceae Acidipila 0.09% 0.52% 2.98% 2.90% Acidobacterium 1.97% 11.46% 0.32% 4.82% Edaphobacter 0.12% 5.06% 1.55% 3.66% Granulicella 0.07% 1.29% 0.06% 0.29% Silvibacterium 0.05% 0.96% 1.28% 1.13% Terriglobus 0.01% 1.27% 0% 0.07% Bryobacteraceae Paludibaculum 0.54% 7.19% 0.18% 2.23% Thermoanaerobaculaceae Thermoanaerobaculum 0% 1.14% 0% 0% Actionobacteria Mycobacteriaceae Mycobacterium 1.57% 1.21% 13.48% 14.50% Mycolicibacterium 0.33% 0.33% 0.47% 1.02% Pseudonocardiaceae Labedaea 0.01% 0% 13.37% 0.45% Pseudonocardia 0.01% 0.01% 1.16% 0.35% Streptosporangiaceae Sinosporangium 0.03% 0.01% 1.95% 0.52% Thermomonosporaceae Actinoallomurus 0.44% 0.86% 18.17% 4.61% Actinocorallia 0.09% 0.22% 1.79% 1.24% Actinomadura 0.12% 0.47% 3.91% 2.06% Bacteriodetes Chitinophagaceae Flavitalea 0.02% 3.27% 0.01% 0.11% Niasterlla 0% 3.57% 0% 0% Puia 0.44% 1.54% 0.43% 2.13% Flavobacteriaceae Flavobacterium 4.44% 0% 0% 0% Sphingobacteriaceae Mucilaginibacter 26.19% 1.79% 0.33% 0.91% Pedobacter 2.92% 0% 0% 0% Sphingobacterium 8.56% 0% 0% 0% Forests 2021, 12, 758 8 of 16

Table 4. Cont.

Phylum Family Genus B_Shiro+ B_Shiro− Y_Shiro+ Y_Shiro− Chloroﬂexi Dictyobacteraceae Dictyobacter 0.34% 0.15% 8.63% 2.88% Thermosporotrichaceae Thermosporothrix 0% 0% 0.61% 2.57% Planctomycetes Tepidisphaeraceae Tepidisphaera 0.17% 1.01% 0% 0.20% Proteobacteria Caulobacteraceae Caulobacter 0.61% 0.67% 0.09% 1.43% Phenylobacterium 0.26% 1.39% 0.40% 1.82% Micropepsaceae Rhizomicrobium 0.56% 7.49% 0.30% 1.48% Bradyrhizobiaceae Bradyrhizobium 0.93% 6.75% 2.88% 2.78% Acetobacteraceae Rhodopila 0.14% 0.90% 3.58% 0.70% Stella 0.39% 0.93% 1.57% 4.24% Rhodospirillaceae Aliidongia 0.40% 0.61% 1.98% 0.79% Sphingomonadaceae Novosphingobium 1.60% 0% 0% 0% Burkholderiaceae Caballeronia 0.50% 0.03% 2.18% 0.61% Paraburkholderia 2.13% 0.61% 1.05% 0.84% Oxalobacteraceae Janthinobacterium 21.94% 0% 0% 0% Forests 2021, 12, x FOR PEER REVIEW 9 of 17 Granulosicoccaceae Sulfuriﬂexus 0.40% 1.01% 0.79% 2.47% Sinobacteraceae Povalibacter 0.86% 1.15% 0.31% 1.38% Pseudomonadaceae Pseudomonas 6.73% 0% 0% 0% Xanthromonadaceae Stenotrophomonas 1.76% 0% 0% 0% Ascomycota were observed. The four classes that were commonly detected in all soil sam- Terrimicrobium 0.30% 0.25% 3.81% 7.29% ples were Dothideomycetes, Eurotinomycetes, Leotiomycetes, and Sordariomycetes (Fig- ure 4b). A total of nine classes from phylum Basidiomycota were observed in all samples, and3.4. three Relative classes Abundance were only of commonly Fungal Communities detected: Agaricomycetes, Geminibasidiomycetes, and TremellomycetesFungal taxonomic (Figure composition 4c). The analysis class that for fouroccurred sample the soils most was frequently conducted was at the Agaricomycetesphylum level in (Figure all the4 samples,a). A total and of sixthis phyla class had were high detected relative in abundance all samples. in Ascomycota, the shiro+ soilsBasidiomycota, compared with Mortierellomycota, the shiro− soils of both and Mucoromycotasites. At the level were of phylum commonly Mucoromycota, observed in all onlysoil Umbelopsidomycetes samples. Basidiomycota was andobserved Mucoromycota in all soil phylasamples were (Figure more 4d). dominant In addition, in the shiro+the classessoils of than unidentified the shiro −Chytridiomycotasoils of both sites. of phylum In contrast, Chytridiomycota, Ascomycota and and Mortierellomycota Mortierellomy- ceteshad of aphylum higher abundanceMortierellomycota in the shiro were− detecsoils comparedted in very with low theproportions shiro+ soils (Figure of both 4e). sites.

FigureFigure 4. Taxonomic 4. Taxonomic composition composition analysis analysis of fu ofngal fungal communities communities at phylum at phylum level level (a) (anda) and class class level level (b– (eb) –inhibitinge) inhibiting the the TricholomaTricholoma matsutake matsutake productionproduction (shiro+) (shiro+) and and nonproduction nonproduction (shiro (shiro−−) soils) soils of of Bonghwa Bonghwa and and Yangyang Yangyang sampling sites.sites. ((bb)) ten ten fungalfungal classesclasses of of Ascomycota Ascomycota phylum, phylum, (c ()c 8) 8 fungal fungal classes classes of of Basidiomycota Basidiomycota phylum, phylum, (d )(d 4) fungal 4 fungal classes classes of Mucoromycotaof Mucoro- mycotaphylum, phylum, and (ande) fungal (e) fungal classes classes from from the phyla the phyla Chytridiomycota, Chytridiomycota, Rozellomycota, Rozellomycota, and Mortierellomycota.and Mortierellomycota.

In the total 168 genera of fungi, 24 genera were observed at a higher than 1% relative abundance (Table 5). Especially one genus, Tricholoma was highly detected in the shiro+ soils (Bonghwa, 63.73%; Yangyang, 68.86%) compared with the shiro− soils (Bonghwa, 0.08%; Yangyang 29.46%) at the two sampling sites. In the Tricholoma genus of the shiro− soil at the Yangyang site, it was revealed that T. saponaceum was detected at 29.45% relative abundance, while T. matsutake was only detected at 0.01% (Table S1). In addition, Um- belopsis from Mucoromycota phylum was commonly observed in all soil samples. It showed relatively high proportions in the shiro+ soil (31.16%) compared with the shiro− soil (1.21%) at the Bonghwa site, while it was more abundantly detected in the shiro− soil (33.51%) than the shiro+ soil (19.62%) of the Yangyang site. The genera Amphinema (16.99%), Astraeus (1.46%), and Sobacina (4.59%) from Basidiomycota phylum were only detected in the shiro− soil at the Bonghwa site. The genera of Hydnum, Cladophialophora, unidentified Herpotrichiellaceae, unidentified Chaetothyriales, Penicillium, unidentified Hyaloscyphaceae, Oidiodendron, unidentified Helotiales, unidentified Ascomycota, and Mortierella were more abundantly detected in the shiro− soils than the shiro+ soils at the two sites. In the fungal genus, ectomycorrhizal (ECM) assemblages and other fungi assemblage were separated. The assemblages of the seven genera of ECM, and one genus of the symbiotic fungal genera, Tricholoma, Tylospora, Astraeus, Sistotrema, Russula, Sebacina,

Forests 2021, 12, 758 9 of 16

The relative abundance of the class level for Ascomycota and Basidiomycota, the most frequently detected phyla in all samples, showed distinct differences between the production/nonproduction soils of T. matsutake. Of the total 29 classes, 10 classes from Ascomycota were observed. The four classes that were commonly detected in all soil samples were Dothideomycetes, Eurotinomycetes, Leotiomycetes, and Sordariomycetes (Figure4b ). A total of nine classes from phylum Basidiomycota were observed in all samples, and three classes were only commonly detected: Agaricomycetes, Geminibasid- iomycetes, and Tremellomycetes (Figure4c). The class that occurred the most frequently was Agaricomycetes in all the samples, and this class had high relative abundance in the shiro+ soils compared with the shiro− soils of both sites. At the level of phylum Mucoromycota, only Umbelopsidomycetes was observed in all soil samples (Figure4d). In addition, the classes of unidentified Chytridiomycota of phylum Chytridiomycota, and Mortierellomycetes of phylum Mortierellomycota were detected in very low proportions (Figure4e). In the total 168 genera of fungi, 24 genera were observed at a higher than 1% relative abundance (Table5). Especially one genus, Tricholoma was highly detected in the shiro+ soils (Bonghwa, 63.73%; Yangyang, 68.86%) compared with the shiro− soils (Bonghwa, 0.08%; Yangyang 29.46%) at the two sampling sites. In the Tricholoma genus of the shiro− soil at the Yangyang site, it was revealed that T. saponaceum was detected at 29.45% relative abundance, while T. matsutake was only detected at 0.01% (Table S1). In addition, Umbelopsis from Mucoromycota phylum was commonly observed in all soil samples. It showed relatively high proportions in the shiro+ soil (31.16%) compared with the shiro− soil (1.21%) at the Bonghwa site, while it was more abundantly detected in the shiro− soil (33.51%) than the shiro+ soil (19.62%) of the Yangyang site. The genera Amphinema (16.99%), Astraeus (1.46%), and Sobacina (4.59%) from Basidiomycota phylum were only detected in the shiro− soil at the Bonghwa site. The genera of Hydnum, Cladophialophora, unidentified Herpotrichiellaceae, unidentified Chaetothyriales, Penicillium, unidentified Hyaloscyphaceae, Oidiodendron, unidentified Helotiales, unidentified Ascomycota, and Mortierella were more abundantly detected in the shiro− soils than the shiro+ soils at the two sites. In the fungal genus, ectomycorrhizal (ECM) assemblages and other fungi assemblage were separated. The assemblages of the seven genera of ECM, and one genus of the symbiotic fungal genera, Tricholoma, Tylospora, Astraeus, Sistotrema, Russula, Sebacina, Tomentella, and Oidiodendron were more abundantly observed in the shiro− soils than the shiro+ soils at the two sites (B_shiro+, 65.91%; B_shiro−, 20.15%; Y_shiro+, 74.43%; Y_shiro−, 37.62%). The relative abundances of other fungal genera assemblages including unknown genera were higher in the shiro− soils than the shiro+ soils at the two sites (B_shiro+, 34.00%; B_shiro−, 74.20%; Y_shiro+, 25.42%; Y_shiro−, 61.74%).

3.5. Similarity of Bacterial and Fungal Communities within/across Sampling Sites The similarity in the bacterial and fungal communities within/across the sampling sites is shown in Table6. The highest similarity index of the bacterial community was observed between the shiro+ soil in Bonghwa and the shiro− soil in Yangyang (0.769), and the lowest value was observed between the shiro− soil in Bonghwa and the shiro+ soil in Yangyang (0.565), or the shiro− soils in Bonghwa and Yangyang (0.565). In the similarity indices of the fungal community, the highest value was observed between the shiro− soils in Bonghwa and Yangyang (0.666), or between the shiro+ soil and the shiro− soil in Yangyang (0.666). The lowest value was observed between the shiro+ soil in Bonghwa and the shiro+ soil in Yangyang (0.578). In fungal communities separated from ECM and other fungi assemblages except for unknown fungi, the similarity of ECM communities was shown to be the highest value in the shiro− soils of the two sites (0.857), whereas low similarity indices were observed between the shiro+ soil and the shiro− soil at the Bonghwa site, the shiro+ soils of two sites, or the shiro+ soil at the Bonghwa site and the shiro− soil at the Yangyang site (Table S2). For other fungal assemblages, the highest Forests 2021, 12, 758 10 of 16

similarity was observed between the shiro− soils of the two sites (0.889), and between the shiro− soil at the Bonghwa site and the shiro+ soil at the Yangyang site (0.889).

Table 5. List of fungal genera inhabiting the Tricholoma matsutake production (shiro+) and nonproduction (shiro−) soils of Bonghwa and Yangyang sampling sites. The fungal genera present more than 1% of at least one sample among the four soil samples.

Phylum Family Genus B_Shiro+ B_Shiro− Y_Shiro+ Y_Shiro− Basidiomycota Tricholomataceae Tricholoma 63.73% 0.08% 68.86% 29.46% Atheliales Amphinema 0% 16.99% 0% 0% Tylospora 0.01% 10.55% 0% 0% Astraeaceae Astraeus 0% 1.46% 0% 0% Cantharellales Sistotrema 0% 0% 2.36% 0.11% Hydnaceae Hydnum 0.01% 0.18% 0% 1.58% Russulaceae Russula 2.04% 0.30% 0% 0.25% Sebacinaceae Sebacina 0% 4.59% 0% 0% Thelephiraceae Tomentella 0.02% 1.54% 0% 0% unidentified 0.03% 1.43% 0.26% 0.23% unidentified unidentified 0.02% 0% 1.34% 1.85% Trimorphomycetaceae Saitozyma 0% 2.10% 0.01% 0.02% Ascomycota Herpotrichiellaceae Cladophialophora 0.08% 7.26% 0.08% 0.85% unidentified 0.04% 3.40% 0.01% 1.73% unidentified unidentified 0% 0.98% 0.17% 3.84% Aspergillaceae Penicillium 0% 1.34% 0.01% 0.21% Hyaloscyphaceae unidentified 0.04% 1.49% 0.18% 1.13% Myxotrichaceae Oidiodendron 0.10% 1.64% 2.38% 7.79% unidentified unidentified 0.03% 1.52% 0.64% 1.14% unidentified unidentified 0.03% 1.81% 0% 0.16% Mortierellomycota Mortierellaceae Mortierella 0.01% 5.48% 0.11% 4.11% Mucoromycota Mucoromycotina Bifiguratus 0.13% 0% 0.70% 1.65% Umbelosidaceae Umbelopsis 31.16% 1.21% 19.62% 33.51% unidentified unidentified unidentified 0.04% 18.19% 0.30% 2.74%

Table 6. The results of Sorensen’s classic similarity index of bacterial and fungal communities within/across sampling sites.

B_Shiro+ B_Shiro− Y_Shiro+ Y_Shiro− Bacterial community B_Shiro+ 1 0.702 0.6 0.769 B_Shiro− 1 0.565 0.565 Y_Shiro+ 1 0.625 Y_Shiro− 1 Fungal community B_Shiro+ 1 0.648 0.578 0.615 B_Shiro− 1 0.628 0.666 Y_Shiro+ 1 0.666 Y_Shiro− 1

4. Discussion Tricholoma matsutake forms in the shiro, as unique and massive aggregates of mycorrhizal hyphae, host plant roots, and soil particles [4,27]. As fruiting bodies form in natural conditions, an understanding of the environment near the fairy ring is crucial to understanding the ecology of T. matsutake [28]. Many studies have shown that the biological and physiochemical characteristics are different between the fairy ring and the adjacent soil, and between the positions within fairy rings likely due to the effects of T. matsutake hyphae [8,13,29–31]. As a biotic environment in fairy rings, various co-existing microbial communities may inﬂuence T. matsutake occurrence in different ways [8,11,13]. Forests 2021, 12, 758 11 of 16

4.1. Distinct Bacterial Community Structure In the work reported here, we compared the difference in the microbial diversity and community between the presence and absence of the shiro (fairy rings) soils, and between two main production regions (Bonghwa and Yangyang) of T. matsutake in Korea. We found that both bacterial and fungal diversity was lower in the shiro+ soil than in the shiro− soil at both sites (Tables2 and3). For microbial diversity inhabiting the fairy ring, bacterial and fungal diversity was signiﬁcantly lower in the T. matsutake-dominant soil compared with the T. matsutake minor soil [11]. The bacterial communities in the active mycorrhizal zone of T. matsutake were much simpler than those at locations far away from the shiro [29]. Moreover, the results of metagenomics analysis showed that the fairy ring zone of T. matsutake had the lowest OTUs, bacterial diversity, and evenness in all sampling zones [10]. In the taxonomic analysis of the bacterial community, our results are in part consistent with a previous study by Oh et al. [11], who documented that Acidobacteria and Proteobacteria were signiﬁcantly higher in the Tm-minor soil. These phyla are abundant in soil environments, but low richness in the Tm-dominant soil could have a negative effect on T. matsutake, such as the competition for resources or secretion of antibiotics to exclude bacteria [32–34]. We found that Acidobacteria was more abundant in the shiro− soil than in the shiro+ soil of both sites. However, there is a large proportion of the phylum of Proteobacteria in the shiro+ soil at the Bonghwa site compared with the shiro− soil, whereas, at the Yangyang site, the phylum showed a higher abundance in the shiro− soil than in the shiro+ soil. Moreover, the Actinobacteria community showed the highest abundance in the shiro+ soil compared with the shiro− soil at the Yangyang site, whereas there was no difference between the shiro+ and shiro− soils at the Bonghwa site. Kim et al. [13] showed that Actinobacteria have a high abundance beneath the fairy ring, while some studies suggested that this community was negatively correlated with the activity of T. matsutake [6,8].

4.2. Distinct Fungal Community Structure In this study, the total amounts of fungal OTUs richness of the shiro− soils were, respectively, approximately six (Bonghwa site) and two times (Yangyang site) higher than those of shiro+ soils (Table3). Our results corroborate previous studies that investigated the decrease in fungal populations in the fairy ring zone of T. matsutake. The total numbers of OTUs and fungal taxa inside and outside the fairy ring zone were higher than those of the fairy ring zone, and the Tm-dominant soil had low fungal richness [9–11]. There are some observations that the sites of occurrence of T. matsutake have low fungal diversity, suggesting that the mycelia of T. matsutake form fruiting bodies under little competition with other microorganisms, and/or T. matsutake can secrete antifungal compounds to exclude other fungal species, promoting its own ﬁtness by reducing competitors [10–12,35]. In our results of the fungal composition at the phylum level, Basidiomycota showed the greatest proportions in all samples, and it also showed higher relative abundances in the shiro+ soils than in the shiro− soils of both sites, whereas Ascomycota showed high abundance in the shiro− soils compared with the shiro+ soils at both sites. There are two previous studies that are consistent with our results about the dominant class in the fairy ring zone. In one study, Lian et al. [12], who compared fungal communities inside, beneath, and outside the fairy ring zone of T. matsutake, also frequently observed Agricomycetes from phylum Basidiomycota beneath the fairy ring zone. In another study, Buée et al. [36] found that Agaricomycetes was the dominant fungal class in forest soil. However, Oh et al. [11] found that Basidiomycota in OTU richness was signiﬁcantly higher in the Tm-minor soils than in the Tm-dominant soils. They suggested that the reduction in fungal richness in the Tm-dominant soils may be caused by the dominance of T. matsutake. The results of the differentiation of the phylum Ascomycota community in all samples were consistent with the previous reports by Kim et al. [10] showing that the classes of Dothideomycetes, Leotiomycetes, and Sordariomycetes from phyla Ascomycota showed higher proportions inside and outside the fairy ring than those in the fairy ring zone. In addition to phylum Forests 2021, 12, 758 12 of 16

Ascomycota, the phylum with the highest abundance was Mucoromycota in all samples. In the phylum Mucoromycota, Umbelopsis was the most abundantly detected from the shiro+ soil at the Bonghwa site as well as the shiro− soil at the Yangyang site. In the previous study, Umbelopsis was frequently detected from the fruiting body and the fairy ring of T. matsutake [28,37], and Oh et al. [11] suggested that Umbelopsis may have positive interactions with T. matsutake. However, there were some differences between the results of the previous study and those obtained in our study because it was also highly detected in the shiro− soil at the Yangyang site. From our results, it can be assumed that Umbelopsis is not necessarily positively correlated with the occurrence of T. matsutake in the shiro− present in soils. In our results of the fungal communities, we separated two main assemblages, the ECM and symbiotic fungal assemblages, and another fungal assemblage. The ECM fungi of generas Tylospora, Astraeus, Sistotrema, Russula, Sebacina, and Tomentella thatwere also detected in a coastal pine forest in the eastern region of Korea, were abundantly detected in the shiro− soil at the Bonghwa site [38–41]. In other fungi assemblages, there are a few fungal genera in the form of saprotrophic fungi groups such as Cladophialophora, penicillium, unidentiﬁed Hyaloscyphaceae, and Mortierella [42]. The fungal communities, except for Tricholoma and other ECMs, have lower proportions in the shiro+ soils than those in the shiro− soils at the two sites (B_shiro+. 34.00%; B_shiro−, 74.20%; Y_shiro+, 25.42%; Y_shiro−, 61.74%). According to the report by Kujawska et al. [42], the fungal communities in the soil of forests could be divided into ECM, saprotrophic, pathotrophic, and other fungi assemblages from different trophic groups. They suggested that the fungal communities in forest soils were closely related to different trophic groups, and were similar in abundance and diversity. Consequently, we found a distinguishable compositional pattern in ECM, and other fungi from the shiro+ soils and the shiro− soils at the two sites.

4.3. Differences between Microbial Communities We observed that the similarities of bacterial and fungal communities were relatively low within/across the sampling sites (Table6). Before we started this study, it was initially expected that the microbial communities between the shiro+ soils of the two sampling sites would be similar to each other. It is well known that T. matsutake had inhibitory effects on soil bacteria and fungi, and it could eliminate the competition for their colonization, the growth of hyphae, and the formation of fruiting bodies. Unexpectedly, our results indicated that the similarity in the bacterial communities between the shiro+ soil of Bonghwa and the shiro− soil of Yangyang showed the highest similarity, whereas the lowest similarity was observed between the shiro− soil of Bonghwa and the shiro+ soil, or the shiro− soil of Yangyang. In addition, the fungal community also differed within/across the samples and sites. The fungal communities between the shiro+ soils at the Bonghwa and Yangyang sites have the lowest community similarity among the samples. However, this could be divided into two main fungal assemblages. For one assemblage of ECM except for unknown fungi, the similarity of ECM communities showed the highest value in the shiro− soils at the two sites (0.857). In a previous study by Kujawska et al. [42], the re-assembly of the soil fungal community at the trophic level could be a strong stochastic component to overcome a general reduction in the similarity of the community composition between different regions. Overall, the simplest explanations for our results showing low similarity between microbial communities within/across the site are as follows. First, in this study, soil samples containing shiro+ and shiro− were only collected once from each site, which was insufﬁcient to compare microbial communities between each site. This was different from sampling many points without favorable permission from farmers. In Korea, a stranger entering a mountain has generally been taboo among villagers of rural communities due to the fear it will result in a bad harvest for T. matsutake that year. Second, the annual mean temperature and annual precipitation of the Yangyang site were slightly higher than those of the Bonghwa site. The changes in climate related to temperature and precipita- Forests 2021, 12, 758 13 of 16

tion influence the changes of microbial communities because the climate changes lead to consequences for the changes of plant communities [43,44]. Ectomycorrhizal fungal species, which are host-dependent symbiotic fungi, are also closely related to changes in climate [44,45]. T. matsutake and truffle, which are ectomycorrhizal mushrooms with high ecological and economic values, are the most sensitive to changes in environmental conditions. Yang et al. [46] reported that high temperature and high precipitation in August were correlated with the high productivity of fruiting bodies. Cejka et al. [44] demonstrated that precipitation is the most important factor for truffle production with drought events reducing truffle yields. We assumed that if climate changes caused changes in vegetation and microbial communities, and changes in vegetation and microbial communities are directly or indirectly affected by the formation of fruiting bodies of T. matsutake, the microbial community may also differ even if there are the same shiro+ soils in different regions. Third, an important environmental factor—soil properties—influence the microbial community. It is well known that the soil environment adjacent to T. matsutake had a lower organic content, and a higher CEC content [10,47], but there is still a large amount of controversy about whether T. matsutake prefers soils containing a low organic matter content or an organic soil content changed by T. matsutake [10]. Although the relationship between the formation of fruiting bodies of T. matsutake and soil properties is still unknown, the impact on microbial communities, diversity, and relative abundance is positively correlated with soil properties [48–50]. Moreover, the report by Kujawska et al. [42] showed that fungal communities differed from trophic groups, soil pH significantly influenced ectomycorrhizal fungal communities, and the volume of coarse woody debris and soil nitrate concentration influenced the saprotrophic fungi community [42]. However, the climate factors and soil properties of the two sites were not measured in this study. Fourth, this study investigated overall bacterial and fungal communities in the production (shiro+) and nonproduction (shiro−) soils of T. matsutake using the Illumina sequencing method. This technology has the advantage of acquiring high-throughput data more quickly for the assessment of microbial communities although there was a limitation in that it produced short read lengths of sequence [10,11,13]. To achieve the accurate assessment of microbial communities, it may be necessary to combine the massive throughput of the next-generation sequencer with the long read lengths by electrophoresis-based methods in Sanger sequencing [10,51]. Nevertheless, it is clear that our results may provide important information to contribute toward an expanding foundation for knowledge on the microbial community in habitats of T. matsutake. Based on the results of this study, further studies of both the microbial communities and abiotic factors in various sites and regions are needed. Furthermore, we expect that it will be the cornerstone for identifying differences in microbial community structure among T. matsutake production soils within/across sites.

5. Conclusions We compared bacterial and fungal communities in T. matsutake production (shiro+) and nonproduction (shiro−) soils in two different regions using the Illumina Miseq sequencing platform. The shiro+ soils showed less OTUs and lower bacterial and fungal diversity than the shiro− soils. The similarity within the microbial communities of shiro+ samples was not signiﬁcant. However, the similarity of fungal communities was affected by their trophic assembly. This suggested that abiotic and biotic factors are important factors that can determine not only the richness of the microbial community but also the quality of the microbial community structure. Further studies are needed to incorporate more diverse samples collected from multiple sites in different seasons. In addition, abiotic factors, such as soil characters, temperature, and humidity should also be synthetically considered. Therefore, the similarity between microbial communities may be due to the fact that the microbial communities in the T. matsutake-dominant soils are closely associated with abiotic factors and biotic factors. Our study may contribute to future studies where the number of study sites is sufﬁcient for understanding the traits of microbial community structures in T. matsutake production soils. Forests 2021, 12, 758 14 of 16

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/f12060758/s1, Table S1: List of fungal species inhibiting the Tricholoma matsutake production (shiro+) and nonproduction (shiro−) soils of Bonghwa and Yangyang sampling sites. The fungal species present more than 1% of at least one sample among the four soil samples, Table S2: The results of Sorensen classic similarity index of fungal communities separating ECM and other fungi assemblages within/across sampling sites. Author Contributions: Conceptualization, G.-H.A., J.-G.H. and J.-H.C.; methodology, G.-H.A., J.- G.H. and O.-T.K.; software, G.-H.A.; validation, G.-H.A., J.-G.H. and J.-H.C.; formal analysis, G.-H.A. and J.-G.H.; investigation, G.-H.A., J.-G.H. and J.-H.C.; resources, G.-H.A.; data curation, G.-H.A.; writing—original draft preparation, G.-H.A. and, J.-G.H.; writing—review and editing, J.-G.H. and O.-T.K.; visualization, G.-H.A. and J.-G.H.; supervision, O.-T.K.; project administration, J.-G.H. and J.-H.C.; funding acquisition, J.-G.H. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Rural Development Administration, Republic of Korea (grant number PJ014766012021). Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors are grateful for the support provided by the National Institute of Horticultural and Herbal Science, Rural Development Administration, Republic of Korea. We also express our sincere gratitude to the officials of Yangyang-gun Agricultural Technology Center and Jae-Mo Sung for their support of our sampling site surveys. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Ogawa, M. The Biology of Matustake Mushroom; Tshkiji Shokkan: Tokyo, Japan, 1978; p. 326. 2. Yamada, A.; Maeda, K.; Ohmasa, M. Ectomycorrhiza formation of Tricholoma matsutake isolates on seedlings of Pinus densiflora in vitro. Mycoscience 1999, 40, 455–463. [CrossRef] 3. Kawai, M.; Terada, O. Artificial reproduction of Tricholoma matsutake (S. Ito et Imai) Sing. II. Effects of vitamins, nucleic acid-related substances, phytohormones and metal ions in the media on the vegetative growth of T. matsutake. Trans. Mycol. Soc. Jpn. 1976, 17, 168–174. 4. Ogawa, M.; Umehara, T.; Kontani, S.; Yamaji, K. Cultivating method of the mycorrhizal fungus, Tricholoma matsutake (Ito et Imai) Sing. (1) Growing method of pine saplings infected with T. matsutake in the field. J. Jpn. For. Soc. 1978, 60, 119–128. [CrossRef] 5. Kawagishi, H.; Hamajima, K.; Takanami, R.; Nakamura, T.; Sato, Y.; Akiyama, Y.; Akiyama, Y.; Sano, M.; Tanaka, O. Growth promotion of mycelia of the matsutake mushroom Tricholoma matsutake by D-isoleucine. Biosci. Biotechnol. Biochem. 2004, 68, 2405–2407. [CrossRef] 6. Vaario, L.M.; Fritze, H.; Spetz, P.; Heinonsalo, J.; Hanajik, P.; Pennanen, T. Tricholoma matsutake dominates diverse microbial communities in different forest soils. Appl. Environ. Microbiol. 2011, 77, 8523–8531. [CrossRef][PubMed] 7. Smith, S.E.; Read, D.J. Mycorrhizal Symbiosis, 2nd ed.; Academic Press: London, UK, 1997. 8. Ohara, H.; Hamada, M. Disappearance of bacteria from the zone of active mycorrhizas in Tricholoma matsutake (S. Ito et Imai) Singer. Nature 1967, 213, 528–529. [CrossRef] 9. Song, H.; Min, K. Microfungal flora of Tricholoma matsutake producing and nonproducing sites in the forest of Pinus densiflora. Korean J. Mycol. 1991, 19, 109–119. 10. Kim, M.; Yoon, H.J.; You, Y.H.; Kim, Y.E.; Woo, J.R.; Seo, Y.G.; Lee, G.M.; Kim, Y.J.; Kong, W.S.; Kim, J.G. Metagenomic analysis of fungal communities inhabiting the fairy ring zone of Tricholoma matsutake. J. Microbiol. Biotechnol. 2013, 23, 1347–1356. [CrossRef] 11. Oh, S.Y.; Fong, J.J.; Park, M.S.; Lim, Y.W. Distinctive feature of microbial communities and bacterial functional profiles in Tricholoma matsutake dominant soil. PLoS ONE. 2016, 11, e0168573. [CrossRef][PubMed] 12. Lian, C.; Narimatsu, M.; Nara, K.; Hogetsu, T. Tricholoma matsutake in a natural Pinus densiflora forest: Correspondence between above- and below-ground genets, association with multiple host trees and alteration of existing ectomycorrhizal communities. New Phytol. 2006, 171, 825–836. [CrossRef] 13. Kim, M.; Yoon, H.; Kim, Y.E.; Kim, Y.J.; Kong, W.S.; Kim, J.G. Comparative analysis of bacterial diversity and communities inhabiting the fairy ring of Tricholoma matsutake by barcoded pyrosequencing. J. Appl. Micro 2014, 117, 699–710. [CrossRef] 14. Frey-Klett, P.; Garbaye, J.; Tarkka, M. The mycorrhiza helper bacteria revisited. New Phytol. 2007, 176, 22–36. [CrossRef][PubMed] 15. Linderman, R.G. Mycorrhizal interactions with the rhizosphere microflora: The mycorrhizosphere effect. Phytopathology 1988, 78, 366–371. 16. Garbaye, J. Helper bacteria: A new dimension to the mycorrhizal symbiosis. New Phytol. 1994, 128, 197–210. [CrossRef] Forests 2021, 12, 758 15 of 16

17. Vivas, A.; Azon, R.; Biro, B.; Barea, J.M.; Ruiz-Lozano, J.M. Influence of bacterial strains isolated from lead-polluted soil and their interactions with arbuscular mycorrhizae on the growth of Trifolium pretense L. under lead toxicity. Can. J. Microbiol. 2003, 49, 577–588. [CrossRef][PubMed] 18. Kim, C.S.; Nam, J.W.; Jo, J.W.; Kim, S.Y.; Han, J.G.; Hyun, M.W.; Sung, G.H.; Han, S.K. Studies on seasonal dynamics of soil-higher fungal communities in Mongolian oak-dominant Gwangneung forest in Korea. J. Mcirobiol. 2016, 54, 14–22. [CrossRef] 19. Oberauner, L.; Zachow, C.; Lackner, S.; Hogenauer, C.; Smolle, K.H.; Berg, G. The ignored diversity: Complex bacterial communities in intensive care units revealed by 16S pyrosequencing. Sci. Rep. 2013, 3, 1413. [CrossRef] 20. Uroz, S.; Ioannidis, P.; Lengelle, J.; Cebron, A.; Morin, E.; Buee, M.; Martin, F. Functional assay and metagenomics analyses reveals differences between the microbial communities inhabiting the soil horizons of a Norway spruce plantation. PLoS ONE 2013, 8, e55925. [CrossRef][PubMed] 21. Kozich, J.J.; Westcott, S.L.; Baxter, N.T.; Highlander, S.K.; Schloss, P.D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the Miseq Illumina sequencing platform. Appl. Environ. Microbiol. 2013, 79, 5112–5120. [CrossRef] 22. Li, W.; Fu, L.; Niu, B.; Wu, S.; Wooley, J. Ultrafast clustering algorithms for metagenomics sequence analysis. Brief. Bioinform. 2012, 13, 656–668. [CrossRef] 23. Cole, J.R.; Wang, Q.; Fish, J.A.; Chai, B.; McGarrell, D.M.; Sun, Y.; Brown, C.T.; Porras-Alfaro, A.; Kuske, C.R.; Tiedje, J.M. Ribosomal Database Project: Data and tools for high throughput rRNA analysis. Nucleic Acids Res. 2014, 42, 633–642. [CrossRef] 24. Nilsson, R.H.; Larsson, K.H.; Taylor, A.F.S.; Bengtsson-Palme, J.; Jeppesen, T.S.; Schige, L.D.; Kennedy, P.; Picard, K.; Glöckner, F.O.; Tedersoo, L.; et al. The UNITE database for molecular identification of fungi: Handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res. 2018.[CrossRef][PubMed] 25. Caporaso, J.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F.D.; Costello, E.K.; Fierer, N.; Pena, A.G.; Goodrich, J.K.; Gordon, J.I.; et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 2010, 7, 335–336. [CrossRef] 26. Chao, A.; Chazdon, R.L.; Colwell, R.K.; Shen, T.J. A new statistical approach for assessing compositional similarity based on incidence and abundance data. Ecol. Lett. 2005, 8, 148–159. [CrossRef] 27. Hosford, D.; Pliz, D.; Molina, R.; Amaranthus, M. Ecology and Management of the Commercially Harvested American Matsutake; General Technical Report PNW-GTR-412; USDA, Forest Service, Pacific Northwest Research Station: Portland, OR, USA, 1997; p. 66. 28. Oh, S.Y.; Park, M.S.; Cho, H.J.; Lim, Y.W. Diversity and effect of Trichoderma isolated from the roots of Pinus densiflora within the fairy ring of pine mushroom (Tricholoma matsutake). PLoS ONE 2018, 13, d0205900. [CrossRef] 29. Kataoka, K.; Shiro, M.; Okura, R.; Oizumi, K.; Fujita, T.; Sasamori, T.; Tokitoh, N.; Yamada, A.; Tanaka, C.; Yamaguchi, M.; et al. The (oxalate) aluminate complex as an antimicrobial substance protecting the “Shiro” of Tricholoma matsutake from soil microorganisms. Biosci. Biotechnol. Biochem. 2017, 81, 102–111. [CrossRef] 30. Hur, T.C.; Park, H. Dynamics of soil microflora and soil enzymes around the fairy-rings of Tricholoma matsutake. J. Korean For. Soc. 2001, 90, 767–773. 31. Hur, T.C.; Park, H.; Joo, S.H.; Ka, K.H. Dynamic changes of soil physicochemical properties in the fairy-rings of Tricholoma matsutake. J. Korean For. Soc. 2004, 93, 26–34. 32. Yun, W.; Hall, I.R.; Evans, L.A. Ectomycorrhizal fungi with edible fruiting bodies 1. Tricholoma matsutake and related fungi. Econ. Bot. 1997, 51, 311–327. [CrossRef] 33. Janssen, P.H. Identifying the dominant soil bacterial taxa in libraries of 16S rDNA and 16S rRNA genes. Appl. Environ. Microbiol. 2006, 72, 1719–1728. [CrossRef] 34. Fierer, N.; Leff, J.W.; Adams, B.J.; Nielsen, U.N.; Bates, S.T.; Lauber, C.L.; Owens, S.; Gilbert, J.A.; Wall, D.H.; Caporaso, G. Cross-biome metagenomics analyses of soil microbial communities and their functional attributes. Proc. Natl. Acad. Sci. USA 2012, 109, 21390–21395. [CrossRef] 35. Takakura, Y. Tricholoma matsutake fruit bodies secrete hydrogen peroxide as a potent inhibitor of fungal growth. Can. J. Microbiol. 2015, 61, 1–4. [CrossRef][PubMed] 36. Buée, M.; Reich, M.; Murat, C.; Morin, E.; Nilsson, R.H.; Uroz, S.; Martin, F. 454 pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytol. 2009, 184, 449–456. [CrossRef][PubMed] 37. Tominaga, Y. Studies on the life history of Japaneses pine mushroom, Armillaria matsutake Ito et Imai. Bull. Hiroshima Agric. Coll. 1963, 2, 105–145. 38. Marino, E.D.; Scattolin, L.; Bodensteiner, P.; Agerer, R. Sistotrema is a genus with ectomycorrhizal species confirmation of what sequence studies already suggested. Mycol. Prog. 2008, 7, 169–176. [CrossRef] 39. Wang, P.; Zhang, Y.; Mi, F.; Tang, X.; He, X.; Cao, Y.; Liu, C.; Yang, D.; Dong, J.; Zhang, K.; et al. Recent advances in population genetics of ectomycorrhizal mushrooms Russula spp. Mycology 2015, 6, 110–120. [CrossRef][PubMed] 40. Obase, K.; Cha, J.Y.; Lee, J.K.; Lee, S.Y.; Lee, J.H.; Chun, K.W. Ectomycorrhizal fungal communities associated with Pinus thunbergii in the eastern coastal pine forests of Korea. Mycorrhiza 2009, 20, 39–49. [CrossRef] 41. Obase, K.; Lee, J.K.; Lee, S.Y.; Chun, K.W. Diversity and community structure of ectomycorrhizal fungi in Pinus thunbergii in the eastern coastal pine forests of Korea. Mycoscience 2011, 52, 383–391. [CrossRef] Forests 2021, 12, 758 16 of 16

42. Kujawska, M.B.; Rudawska, M.; Wilgan, R.; Leski, T. Similarities and differences among soil fungal assemblages in managed forests and formerly managed forest reserves. Forests 2021, 12, 353. [CrossRef] 43. Waldrop, M.P.; Firestone, M.K. Response of microbial community composition and function to soil climate change. Microb. Ecol. 2005, 52, 716–724. [CrossRef] 44. Guo, Y.; Li, X.; Zhao, Z.; Wei, H.; Gao, B.; Gu, W. Prediction of the potential geographic distribution of the ectomycorrhizal mushroom Tricholoma matsutake under multiple climate change scenarios. Sci. Rep. 2017, 7, 46221. [CrossRef] 45. Cejka,ˇ T.; Trnka, M.; Krusic, P.J.; Stobbe, U.; Oliach, D.; Václavík, T.; Tegel, W.; Büntgen, U. Predicted climate change will increase the trufﬂe cultivation potential in central Europe. Sci. Rep. 2020, 10, 21281. [CrossRef][PubMed] 46. Yang, X.; Luedeling, E.; Chen, G.; Hyde, K.D.; Yang, Y.; Zhou, D.; Xu, J.; Yang, Y. Climate change effects fruiting of the prize matsutake mushroom in China. Fungal Divers. 2012, 56, 189–198. [CrossRef] 47. Stucki, J.W.; Gan, H.; Wilkinson, H.T. Effects of microorganisms on phyllosilicate properties and behavior. In Interacting Processes in Soil Science; Wagenet, R.J., Baveye, P., Stewart, B.A., Eds.; Advances in Soil Science; CRC Press: Boca Raton, FL, USA, 1992; pp. 227–251. 48. Jones, R.T.; Robeson, M.S.; Lauber, C.L.; Hamady, M.; Knight, R.; Fierer, N. A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. ISME J. 2009, 3, 442–453. [CrossRef][PubMed] 49. Lauber, C.L.; Hamady, M.; Knight, R.; Fierer, N. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl. Environ. Microbiol. 2009, 75, 5111–5120. [CrossRef][PubMed] 50. Rousk, J.; Baath, E.; Brookes, P.C.; Lauber, C.L.; Lozupone, C.; Caproaso, J.G.; Knight, R.; Fierer, N. Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME J. 2010, 4, 1340–1351. [CrossRef] 51. Hert, D.G.; Fredlake, C.P.; Barron, A.E. Advantages and limitations of next-generation sequencing technologies: A comparison of electrophoresis and non-electrophoresis methods. Electrophoresis 2008, 29, 4618–4626. [CrossRef]